A method to increase reproducibility in adult ventricular myocyte sizing and flow cytometry: Avoiding cell size bias in single cell preparations.

RATIONALE:Flow cytometry (FCM) of ventricular myocytes (VMs) is an emerging technology in adult cardiac research that is challenged by the wide variety of VM shapes and sizes. Cellular variability and cytometer flow cell size can affect cytometer performance. These two factors of variance limit assay validity and reproducibility across laboratories. Washing and filtering of ventricular cells in suspension are routinely done to prevent cell clumping and minimize data variability without the appropriate standardization. We hypothesize that washing and filtering arbitrarily biases towards sampling smaller VMs than what actually exist in the adult heart. OBJECTIVE:To determine the impact of washing and filtering on adult ventricular cells for cell sizing and FCM. METHODS AND RESULTS:Left ventricular cardiac cells in single-cell suspension were harvested from New Zealand White rabbits and fixed prior to analysis. Each ventricular sample was aliquoted before washing or filtering through a 40, 70, 100 or 200μm mesh. The outcomes of the study are VM volume by Coulter Multisizer and light-scatter signatures by FCM. Data are presented as mean±SD. Myocyte volumes without washing or filtering (NF) served as the "gold standard" within the sample and ranged from 11,017 to 46,926μm3. Filtering each animal sample through a 200μm mesh caused no variation in the post-filtration volume (1.01+0.01 fold vs. NF, n = 4 rabbits, p = 0.999) with an intra-assay coefficient of variation (%CV) of <5% for all 4 samples. Filtering each sample through a 40, 70 or 100μm mesh invariably reduced the post-filtration volume by 41±10%, 9.0±0.8% and 8.8±0.8% respectively (n = 4 rabbits, p<0.0001), and increased the %CV (18% to 1.3%). The high light-scatter signature by FCM, a simple parameter for the identification of ventricular myocytes, was measured after washing and filtering. Washing discarded VMs and filtering cells through a 40 or 100μm mesh reduced larger VM by 46% or 11% respectively (n = 6 from 2 rabbits, p<0.001). CONCLUSION:Washing and filtering VM suspensions through meshes 100μm or less biases myocyte volumes to smaller sizes, excludes larger cells, and increases VM variability. These findings indicate that validity and reproducibility across laboratories can be compromised unless cell preparation is standardized. We propose no wash prior to fixation and a 200μm mesh for filtrations to provide a reproducible standard for VM studies using FCM

Contra-Analysis: Prioritizing Meaningful Effect Size in Scientific Research

At every phase of scientific research, scientists must decide how to allocate limited resources to pursue the research inquiries with the greatest potential. This prioritization dictates which controlled interventions are studied, awarded funding, published, reproduced with repeated experiments, investigated in related contexts, and translated for societal use. There are many factors that influence this decision-making, but interventions with larger effect size are often favored because they exert the greatest influence on the system studied. To inform these decisions, scientists must compare effect size across studies with dissimilar experiment designs to identify the interventions with the largest effect. These studies are often only loosely related in nature, using experiments with a combination of different populations, conditions, timepoints, measurement techniques, and experiment models that measure the same phenomenon with a continuous variable. We name this assessment contra-analysis and propose to use credible intervals of the relative difference in means to compare effect size across studies in a meritocracy between competing interventions. We propose a data visualization, the contra plot, that allows scientists to score and rank effect size between studies that measure the same phenomenon, aid in determining an appropriate threshold for meaningful effect, and perform hypothesis tests to determine which interventions have meaningful effect size. We illustrate the use of contra plots with real biomedical research data. Contra-analysis promotes a practical interpretation of effect size and facilitates the prioritization of scientific research.Comment: 4 figures, 8000 word

Prognostic factors for esophageal squamous cell Carcinoma-A Population-Based study in Golestan province, Iran, a high incidence area

Golestan Province in northern Iran is an area with a high incidence of esophageal squamous cell carcinoma (ESCC). We aimed to investigate prognostic factors for ESCC and survival of cases in Golestan, on which little data were available. We followed-up 426 ESCC cases participating in a population-based case-control study. Data were analyzed using the Kaplan-Meier method and the Cox proportional hazard models. Median survival was 7 months. Age at diagnosis was inversely associated with survival, but the association was disappeared with adjustment for treatment. Residing in urban areas (hazard ratio, HR = 0.70; 95 CI 0.54-0.90) and being of non-Turkmen ethnic groups (HR = 0.76; 95 CI 0.61-0.96) were associated with better prognosis. In contrast to other types of tobacco use, nass (a smokeless tobacco product) chewing was associated with a slightly poorer prognosis even in models adjusted for other factors including stage of disease and treatment (HR = 1.38; 95 CI 0.99-1.92). Opium use was associated with poorer prognosis in crude analyses but not in adjusted models. Almost all of potentially curative treatments were associated with longer survival. Prognosis of ESCC in Golestan is very poor. Easier access to treatment facilities may improve the prognosis of ESCC in Golestan. The observed association between nass chewing and poorer prognosis needs further investigations; this association may suggest a possible role for ingestion of nass constituents in prognosis of ESCC. © 2011 Aghcheli et al

Down-regulation of miR-133a and miR-539 are associated with unfavorable prognosis in patients suffering from osteosarcoma

Background: MicroRNAs (miRNAs) play key roles in cancer development and progression. The purpose of the present study was to determine the expression levels of miR-133a and miR-539 in osteosarcoma patients and to further investigate the clinicopathological, and prognostic value of these miRNAs. Methods: The expression levels of miR-133a and miR-539 were determined by qRT-PCR. Associations between miRNAs expressions and various clinicopathological characteristics were analyzed. Survival rate was determined with Kaplan-Meier and statistically analyzed with the log-rank method between groups. Survival data were evaluated through multivariate Cox regression analysis Results: Our findings revealed that the miR-133a expression was significantly decreased in clinical osteosarcoma tissues compared to adjacent normal bone tissues. The expression level of miR-539 was decreased in clinical osteosarcoma tissues as compared to those adjacent normal tissues. Low expressions of miR-133a and miR-539 were significantly association with advanced TNM stage (P=0.002; P=0.001), and metastasis or recurrence (P=0.001; P=0.01). Furthermore, Kaplan-Meier survival analysis and log-rank test showed that the low expressions of miR-133a and miR-539 were correlated with the reduced overall survival of osteosarcoma patients. Multivariate Cox proportional hazards model showed that decreased expressions of miR-133a and miR-539 (P=0.007; P=0.02), TNM stage (P=0.001; P=0.002), and metastasis or recurrence (P=0.005; P=0.026) were independent prognostic markers of overall survival of patients. Conclusion: These results suggest that decreased miR-133a and miR-539 expressions may participate in the progression of osteosarcoma. Together, these results showed that miR-133a and miR-539 may have their role in both progression and prognosis of osteosarcoma. © 2015 Mirghasemi et al

Down-regulation of microRNA-26a and up-regulation of microRNA-27a contributes to aggressive progression of human osteosarcoma

Background: Osteosarcoma is the most common primary bone malignancy with high local aggressiveness and rapid metastasizing potential, resulting in poor survival. Increasing reports suggest that deregulated microRNAs (miRNAs) might provide novel therapeutic targets for cancers. However, the expression of miR-26a and miR-27a in osteosarcoma need further investigation in clinical samples. In our study, we evaluate the expression of these miRNAs in osteosarcoma tissues and compared with paired adjacent non-tumor bone tissues using RT-qPCR. Methods: Total RNA was purified from patients with osteosarcoma and noncancerous bone tissues. Real-time PCR was applied to quantify the expression level of miR-26a and miR-27a. Moreover, the correlation of these markers with clinicopathological characteristics was also evaluated in osteosarcoma patients. A cox proportional hazards model was performed to assess multivariate analyses of prognostic values. Results: Our result suggested that miR-26aexpression level in osteosarcoma bone tissue was significantly lower than that in the paired noncancerous bone tissues. MiR-27a expression was higher in osteosarcoma bone tissue in comparison with paired noncancerous bone tissues. The results indicated that low expression level of miR-26a and high expression of miR-27a were associated with high TNM stage (P = 0.001; P = 0.012), tumor grade (P = 0.007; P = 0.016), and distant metastasis (P = 0.004; P = 0.001). Kaplan-Meier analysis and log-rank test indicated that patients with low expression of miR-26a and high expression of miR-27a had shorter overall survival (log-rank test: P < 0.001). Multivariate Cox proportional hazards model analysis showed that low expression of miR-26a and high expression of miR-27a (P = 0.021; P = 0.011), high TNM stage (P = 0.001; P = 0.003), tumor grade (P = 0.005; P = 0.01), and distant metastasis.(P = 0.002; P = 0.005) were independent prognostic factors for overall survival patients with osteosarcoma cancer. Conclusions: In conclusion, our findings suggested that expression level of miR-26a and miR-27a contributes to aggressive progression of this malignancy. Therefore, may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients. © 2015 Taheriazam et al

Comparison of normal values of Duplex indices of vertebral arteries in young and elderly adults

<p>Abstract</p> <p>Background</p> <p>Considering the role of aging in brain atrophy and cerebral vascular demand, we carried out this study to clarify the role of aging in duplex indices of vertebral arteries.</p> <p>Methods</p> <p>From June 2005 to June 2006, 96 volunteers with age range of 20 to 95 years, were evaluated with color doppler for duplex indices of vertebral arteries. Sever hemodynamic stenosis was excluded in all of these patients. These volunteers were subdivided in two groups: younger and older than 60 year old. In all of these patients we measured diameter, peak systolic velocity (PSV), resistive index (RI), and flow volume (FV) of vertebral arteries in right and left sides.</p> <p>Results</p> <p>There was no significant difference in diameter, PSV, RI and FV between two groups. We have clarified that in patients younger than 60 year old, comparing right and left vertebral arteries, PSV and FV were higher in left side.</p> <p>Conclusion</p> <p>Duplex indices of vertebral arteries are age independent in adults.</p

Response to dietary supplementation of L-glutamine and L-glutamate in broiler chickens reared at different stocking densities under the hot, humid tropical conditions

A study was conducted to determine whether supplementing AminoGut (a commercial dietary supplement containing a mixture of l-glutamine and l-glutamic acid) to broiler chickens stocked at 2 different densities affected performance, physiological stress responses, foot pad dermatitis incidence, and intestinal morphology and microflora. A randomized design in a factorial arrangement with 4 diets [basal diet, basal diet + 0.5% AminoGut from d 1 to 21, basal diet + 0.5% AminoGut from d 1 to 42, and basal diet + virginiamycin (0.02%) for d 1 to 42] and 2 stocking densities [0.100 m2/bird (23 birds/pen; LD) or 0.067 m2/bird (35 birds/pen; HD)]. Results showed that villi length and crypt depth were not changed by different dietary treatments. However, birds in the HD group had smaller villi (P = 0.03) compared with those of the LD group. Regardless of diet, HD consistently increased the serum concentrations of ceruloplasmin, α-1 acid glycoprotein, ovotransferin, and corticosterone (P = 0.0007), and elevated heterophil to lymphocyte ratio (0.0005). Neither AminoGut supplementation nor stocking density affected cecal microflora counts. In conclusion, under the conditions of this study, dietary supplementation of AminoGut, irrespective of stocking density, had no beneficial effect on growth performance, intestinal morphology, and physiological adaptive responses of broiler chickens raised under hot and humid tropical conditions. However, AminoGut supplementation from d 1 to 42 was beneficial in reducing mortality rate. Also, the increased serum concentrations of a wide range of acute phase proteins together with elevated corticosterone and heterophil to lymphocyte ratio suggested that high stocking density induced an acute phase response either indirectly as a result of increased incidence of inflammatory diseases such as foot pad dermatitis or possibly as a direct physiological response to the stress of high stocking density

Betaine and Isoquinoline Alkaloids Protect against Heat Stress and Colonic Permeability in Growing Pigs

Heat stress (HS) compromises productivity of pork production, in part as a result of increased oxidative stress and inflammatory responses, particularly within the gastrointestinal tract. This study aimed to investigate whether plant-derived betaine and isoquinoline alkaloids could ameliorate HS in pigs. Fifty female Large White × Landrace grower pigs, which were acclimated to control (CON), control plus betaine (BET), or control plus isoquinoline alkaloids (IQA) diets for 14 days were then exposed to heat stress or thermoneutral condition. Both BET and IQA partially ameliorated increases in respiration rate (p = 0.013) and rectal temperature (p = 0.001) associated with HS conditions. Heat stress increased salivary cortisol concentrations and reduced plasma creatinine, lactate, and thyroid hormone concentrations. Heat stress increased colon FD4 permeability, which was reduced by IQA (p = 0.030). Heat stress increased inflammation in the jejunum and ileum, as indicated by elevated interleukin-1β (p = 0.022) in the jejunum and interleukin-1β (p = 0.004) and interleukin-8 (p = 0.001) in the ileum. No differences in plasma total antioxidant capacity (TAC) were observed with HS, but betaine increased plasma TAC compared to IQA. Dietary BET increased betaine concentrations in the jejunum, ileum (p < 0.001 for both), plasma, liver, kidney (p < 0.010 for all), urine (p = 0.002) and tended to be higher in muscle (p = 0.084). Betaine concentration was not influenced by HS, but it tended to be higher in plasma and accumulated in the liver. These data suggest that betaine and isoquinoline alkaloids supplementation ameliorated consequences of heat stress in grower pigs and protected against HS induced increases in colonic permeability